Herpes simplex virus 1 (HSV-1) is a neurotropic virus that infects over 70% of the world adult population. It establishes a lifelong latency in trigemina ganglia and reactivates sporadically to spread to nave population at their young age. It causes a wide range of clinical manifestations such as cold sores, genital ulceration, keratitis, and the deadly herpes encephalitis. Infected cell protein no. 0 (ICP0), an immediate early protein that is a key regulator for both lytic infection and latency reactivation, plays a vital role in the counteractions against host anti-viral defenses. ICP0 contains an E3 ubiquitin ligase, which mediates proteasomal degradation of several host restrictive factors and therefore enhance viral gene expression. It also interacts with numerous cellular proteins in order to regulate a diverse array of cell pathways participating in anti-viral defenses. The multiple functions of ICP0 located in different domains of ICP0 must coordinate with each other to orchestrate a robust infection. Results from our lab and several other labs have shown that a dynamic nuclear domain 10 (ND10) structure undertakes a close interplay with ICP0 in the tug-of-war of HSV-1 infection. On one hand, ND10 converges at viral DNA for gene repression. On the other hand, ICP0 merges with ND10 and degrades its organizer protein PML (promyelocytic leukemia) in order to disperse ND10 bodies and release the repression. We have previously reported that ICP0-ND10 interaction is a dynamic process that includes sequential steps of adhesion, fusion and retention, and specific ICP0 domains are involved in the faithful execution of these dynamic steps. The overall objective of the present application is to dissect the functional domains of ICP0 that are important for ND10 fusion process and E3 ubiquitin ligase activity, and to delineate ICP0 domain coordination in its counteractions against the host defense. We plan to construct ICP0 mutant viruses to identify functional domains, to characterize the coordination among different domains, and to isolate proteins interacting to these specific domains. We intend to pursue the following specific aims in this grant proposal: (i) to delineate the molecular basis of ND10 fusion process and (ii) to characterize the regulatory mechanisms of ICP0 E3 ubiquitin ligase. These studies are highly relevant to the mission of understanding infectious diseases caused by HSV-1. The outcomes of these proposed studies will greatly advance our knowledge of the initiation of HSV-1 infection, as well as the molecular mechanisms of host defenses. This study is expected to have a significant impact in developing new anti-herpes therapies and in understanding cellular defense mechanisms against DNA viruses.